Optimization of Multiplex Real-time PCR Assay for Detection and Quantification of Vibrio spp. and Total Bacteria

Introduction: Vibrio spp. are opportunistic human pathogens that cause gastroenteritis with a high lethality rate. The infection is associated with consumption of seafood or with exposure of contaminated water.

Purpose: This study describes the development of a Multiplex Real-time PCR assay for simultaneous detection and quantification of total bacteria (16S rDNA) and three kinds of Vibrio spp. such as Vibrio vulnificus (vvhA), V. parahaemolyticus (tlh), and V. anguillarum (ToxR).

Methods: For the multiplex assay, 4 sets of primer pairs and probes were newly designed as well as the Real-time PCR cycling protocol, fluorescent detection parameters, and reaction mixture components were optimized.

Results: The optimal conditions of PCR used the following final concentrations for each 800 nM of the ToxR, 200 nM of the 16S rDNA, 50 nM of the vvhA, and tlh forward/reverse primers and probe. The optimal cycling parameters consisted of the initial denaturation at 95°C hold for 1 min followed by 40 cycles of DNA amplification: denaturation step at 95°C for 15 s and combined annealing/extension step at 56°C for 50 s. The species-specific targets showed negative results and 16S rDNA was positive against 28 bacterial species including Vibrio spp. The assay was optimized for the detection of low numbers up to Log 0 CFU/reaction and the R² values were 0.99 for the standard curve of vvhA, tlh, and ToxR and 0.98 for the 16S rDNA.

Significance: The Multiplex Real-time PCR assay could be useful for rapid monitoring of seafood and marine water quality.